We have focused on HIV-1 entry into cells, a process which is initiated by fusion between the viral envelope and the host cell membrane. Interaction of HIV-1 envelope glycoproteins (gp120-gp41) with host cell CD4 and additional co-receptors such as CXCR4 and CCR5, that determine the tropism of different HIV-1 isolates, triggers this fusion cascade. Several viral envelope glycoprotein oligomers assemble into a viral fusion machine which forms a molecular scaffold responsible for bringing the viral membrane close to the target cell membrane, and creating the architecture that enables lipid bilayers to merge. Triggering the fusion machine results in drastic conformational changes in viral envelope glycoproteins. The fusion reaction then undergoes multiple steps before the final event occurs which allows delivery of the nucleocapsid into the cell. Our overall approach to the elucidation of mechanisms of (gp120-gp41)-mediated membrane fusion involves a dissection of steps in the fusion cascade using spectrofluorometry and quantitative fluorescence video microscopy, and analysis of the kinetic intermediates using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques. We have examined mutations in the ectodomain of the HIV-1 transmembrane glycoprotein gp41 within a region immediately adjacent to the membrane-spanning domain for their effect on the outcome of the fusion cascade. Using our recently-developed 3 color assay we have assessed the ability of the mutant gp41's to transfer lipid and small solutes from susceptible target cells to the gp120-gp41-expressing cells. We found an interesting phenotype that was not capable of inducing syncytia formation but still mediated dye transfer indicating that the fusion cascade was blocked beyond the stage of small fusion pore formation. To analyze the biochemical nature of intermediates and identify domains of HIV-1 gp41 which are involved in the operation of the HIV-1 fusion machine we have applied photosensitized labeling. The method is based on activation of a hydrophobic probe Iodo-Naphthyl-Azide(INA), that rapidly partitions into the membrane bilayer of virus and cells, by a membrane-incorporated fluorescent analog. Using this technique, INA labeling of gp41 can be confined to the domains inserted into the target membrane. We have achieved efficient and reproducible preparative scale purification of gp41 in quantities sufficient for analysis by MALDI-MS or microsequencing. In our studies on the role of the target membrane we have obtained evidence that the fusion machine may assemble in glycosphingolipid-enriched microdomains in the target membrane. Evidence for this concept is based on our finding that treatment of susceptible cell with a specific inhibitor of glycosphingolipid biosynthesis affects HIV-1 infection and fusion, and that this activity can be recovered following addition of a purified Globotriaosylceramide to the impaired cells. The implication is that inhibitors of the glycosphingolipid biosynthetic pathway(s) or of glycosphingolipid-mediated interactions of HIV-1 with the cell surface may prevent HIV infection. Z01 BC 08303-28